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Research ArticleAqueous Extract of Clerodendranthus spicatus ExertsProtective Effect on UV-Induced Photoaged Mice Skin

Lan Wang,1 Xie Zhang,2 Yong-xian Li,1 Lie-qiang Xu,3 Cai-lan Li,3 Zhen-biao Zhang,3

Jia-li Liang,3 Zi-ren Su,3 Hui-fang Zeng,1 and Yu-cui Li3

1The First Affiliated Hospital of Chinese Medicine, Guangzhou University of Chinese Medicine, 12 Airport Road,Bai Yun District, Guangzhou 510405, China2School of Chinese Medicine, Faculty of Science, The Chinese University of Hong Kong, Shatin, Hong Kong3School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou Higher Education Mega Center,232 Wai Huan Dong Road, Guangzhou 510006, China

Correspondence should be addressed to Hui-fang Zeng; [email protected] and Yu-cui Li; [email protected]

Received 28 July 2016; Revised 11 September 2016; Accepted 18 September 2016

Academic Editor: Yong C. Boo

Copyright © 2016 Lan Wang et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Clerodendranthus spicatus (Thunb.) C.Y.Wu (CS) is commonly used to treat kidney diseases in traditional Chinese medicine forits prominent anti-inflammatory effect and nourishing function to kidneys. In this study, aqueous extract of CS was assessed forits protective effect on UV-induced skin damage of mice. The chemical compositions of CS aqueous extract were determined byHPLC-ESI-MS/MS, in which 10 components were identified. During the experimental period, CS (0.9, 1.8, and 3.6 g/mL) wasexternally applied to shaved dorsal skins ofmice prior toUV irradiation, daily for tenweeks.The results presented thatCS (3.6 g/mL)apparently improved photodamaged skin appearance such as erythema, edema, and coarseness.The abnormal epidermal thickeningwas significantly reduced, and the dermal structures becamemore complete.Theunderlying protectivemechanismswere associatedwith improving antioxidant enzymes activities including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase(GSH-Px), downregulating inflammatory cytokines (IL-1𝛽, IL-6, TNF-𝛼, COX-2, and PGE

2) expressions, recovering collagen

density, and reducingmatrix metalloproteinases productions. Sun protection factor of CS (3.6 g/mL) was 16.21±0.03. Our findingsfor the first time demonstrated that CS had therapeutic effect on the photoaged skin. The results indicated that CS is a potentialagent for photoprotective cosmetics.

1. Introduction

Long-term exposure to sunlight usually accelerates exoge-nous aging of skin, namely, photoaging. The characters ofphotoaging are laxity, coarse winkles, roughness, and irregu-lar pigmentation [1]. Ultraviolet (UV) irradiation is the mostimportant factor in the process of photoaging. It activates acomplex cascade of biochemical reactions involving oxidativestress and inflammatory reaction and subsequently altersthe structure and function of extracellular matrix (ECM)by degrading collagen and elastic fibers, finally leading toabnormal morphological and histological changes in humanskin [2].

UV includes UVA, UVB, and UVC, with wavelengthsof 320∼400 nm, 280∼320 nm, and 100∼280 nm, respectively.

Among them, both UVB and UVA can cause skin damageand photoaging [3]. UVB, mostly absorbed in the epider-mis, mainly triggers proinflammatory mediators and stim-ulates the expression of matrix metalloproteinases (MMPs),which degrades ECM and ultimately results in wrinklesand sagging appearance [4]. Differing from UVB, UVAreaches the dermis. It generates reactive oxygen species(ROS), induces MMPs synthesis, and causes mutation ofmitochondrial DNA. Therefore, on one hand, excess UVlight could inhibit antioxidant enzymes activities, destroythe antioxidant defense systems, and finally bring aboutoxidative damage in the skin [1]. UV-induced oxidative stressfurther activates MAPK signaling pathway and then inducesAP-1, resulting in increased activation of cytokines suchas interleukins and tumor necrosis factor-𝛼 (TNF-𝛼) [5].

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016, Article ID 9623957, 11 pageshttp://dx.doi.org/10.1155/2016/9623957

2 Evidence-Based Complementary and Alternative Medicine

On the other hand, UV expedites the production of NF-𝜅Band stimulates downstream inflammatory mediators such asinterleukin-1𝛽 (IL-1𝛽), IL-6, and TNF-𝛼.These inflammatorymediators synergistically promote the accumulation of ROS,further augment the expression of MMPs, finally lead toerythema and edema, which are characters of inflammationresponses. What is more, UV also stimulates skin cells toproduce cyclooxygenase-2 (COX-2), catalyzing the synthesisof prostaglandin E2 (PGE

2) from prostanoid precursors and

deteriorating the skin inflammation [6].To summarize, the excessive production of ROS and

inflammatory cytokines is closely linked to the expression ofMMPs and consequently causes coarse and corrugated skin.Upon the background of photoaging, we can speculate thatantioxidant and anti-inflammatory agents have potentialityto delay this premature process [2].

Clerodendranthus spicatus (Thunb.) C.Y.Wu (Orthosiphonstamineus) (CS) is widely cultivated in tropical regions andthe Pacific regions such as China, India, Malaysia, andAustralia. It is a kind of traditional Chinese medicine totreat nephritis, lithangiuria, and cystitis [7, 8]. Based on theplenty researches we found CS contains various bioactivecomponents such as flavonoids, diterpenes, and phenols [9]to exhibit the anti-inflammatory [10], analgesic [11], lipidperoxidation inhibiting [12], and free radical scavenging [13]activities, which are in agreement with mainstream therapiesof containment of the progress of senescence [14]. Whatis more, in traditional Chinese medicine theory, kidneysupports human body to be young and vigorous. Our team[15] and other researchers [16] have reported that CS has goodantiaging effect through improving antioxidant capacity ofaged mice induced by D-galactose. It attracts our attentionto reveal if CS can prevent exogenous senescence. Therefore,this study aimed to investigate whether CS could remedyUV-induced photoaging.

2. Methods and Materials

2.1. Materials and Chemicals. We used CAT, SOD, GSH-Px,MDA, andmouse hydroxyproline (Hyp) assay kits (JianchengInstitute of Biotechnology, Nanjing, China), as well as ELISAkits: TNF-𝛼, IL-6, MMP-1, MMP-3, IL-1𝛽, COX-2 and PGE

2

(Cheng Lin Biological Technology Co., Ltd., Beijing, China).All other reagents used were of analytical grade.

2.2. Preparation of CS Aqueous Extract. CS (lot 20141101) wasgifted by XinXing Tongren Pharmaceutical Co., Ltd. (Yunfu,China). 1 kg of dried plants added to 12-fold distilled waterwas soaked overnight. After being decocted for 2 h twice, theextracted solution was merged and concentrated to 1 L andthen mixed with 2 L 95% ethanol for 24 hours. After that, thesolution was filtered and the ethanol was recycled by rotaryevaporator. The filtered fluid was diluted by distilled water to0.1 L (10 g crude drug/mL) and then yielded to three differentconcentrations: 0.9 g/mL, 1.8 g/mL, and 3.6 g/mL.

2.3. HPLC-ESI-MS/MS Analysis of CS Aqueous Extract.HPLC analysis was executed on a Shimadzu LC-20A HPLC

system consisting of a SPD-M20A PDA detector, a LC-20AD pump, a SIL-20AC automatic sampler, and CTO-20A thermostatic column compartment (Shimadzu Kyoto,Japan). A Kromasil KR100-5C18 column (250mm × 4.6mm,E17096) whose flow rate is 0.4mL/min, column temperatureis 25∘C, and injection volume is 10 𝜇L was used for separa-tion. CS was diluted to the concentration of 1mg/mL withmethanol and filtered through 0.22𝜇m microporous mem-brane for HPLC-electrospray ionization-MS (HPLC-ESI-MS/MS) analysis. The mobile phase included water (solventA) and acetonitrile (solvent B) and was set in gradient mode(0–10min: 5%B→8%B; 10–15min: 8%B→18%B; 15–25min:18%B; 25–40min: 18%B→30%B; 40–50min: 30%B→35%B;50–52min: 35%B→40%B; 52–57min: 40%B→45%B; 57–67min: 45%B→90%B; and 67–77min: 90%B).

ESI source for mass detection was performed via Triple�TOF 5600 system. Data acquisition and process were con-ducted with MultiQuant� Software system (AB SCIEX,USA). Both positive and negative ionization modes of massspectra were acquired. The optional parameters were setas follows: ion spray voltage (−4500 kV); ion source heater(550∘C); curtain gas (N

2, 35 psi); ion source gas 1 (55 psi);

and ion source gas 2 (55 psi). The mass analyzer scan was100 to 1000m/z.The entrance potential (EP) and declusteringpotential (DP) were −10 eV and −100 eV.

2.4. Animals. FemaleKunmingmice (6–8weeks old, 18–22 g)were brought from animal center of Guangzhou Universityof Chinese Medicine (GZUCM, Guangzhou, China). Allexperiments were performed in specific pathogen-free laband approved by the Institutional Animal Care andUseCom-mittee at GZUCM (approval number SCXK (Guangzhou)2014-0085). After acclimating for 1 week,micewere randomlydivided into seven groups (𝑛 = 9). The grouping was asfollows: naıve control (NC), shamcontrol (SC),model control(MC), vehicle control (VC), and three treated groups (CS-low dose, CS-L; CS-middle dose, CS-M; and CS-high dose,CS-H). Experimenters anesthetized mice via inhaling etherand then used shavers to shave mice dorsal skins about 2.5× 3 cm2. Except for NC group, all others were shaved. Hencethe shaving was performed daily.

2.5. Establishing Photoaged Model and Treatment. The spec-trum of UV light and ratio of UVB/UVA were set accord-ing to solar irradiation. Combined UVB and UVA lamps(Waldmann UV800, Germany) emitted the UV light. Tocontrol the UV radiation energy, an electronic controllerwas prepared. The UV lamp was fixed 30 cm above micecages. The radiation energy supplied from the lamps (630× 10 𝜇W) was measured with a UV radiometer (WaldmannLichttechnik GmbH, Germany). The starting intensity of UVlight in the first week was at one minimal erythemal dose(MED), which equaled 100mJ/cm2. The following dose wasincreased by one MED every week until the fourth week.Then, the intensity of irradiation was at 4 MED for theremaining weeks [17]. The UV irradiation frequency wasset at five times a week for ten weeks. Except for NC andSC group, all others groups were irradiated. Prior to being

Evidence-Based Complementary and Alternative Medicine 3

Table 1: Grading scales for evaluation of photoaging.

Grade Evaluation criteria0 No wrinkles; fine striations running the length of the body1 Fine striations2 A few shallow wrinkles; disappearance of all fine striations3 Shallow wrinkles across the dorsal skin4 Deep and coarse wrinkles with laxity5 Increased deep wrinkles6 Severe wrinkles; development of tumors or lesions

irradiated, the treated groups were externally embrocatedwith CS (0.9, 1.8, and 3.6 g/mL) and simultaneously the VCgroup were externally embrocated with distilled water everyday during the whole experimental period.

2.6. Images Records of Dorsal Skins. Experimenters tookphotos of every dopeymouse skinweekly for 10w.Themacro-scopic visual scores were calculated by blinded investigatorsaccording to the grading scale [18] (Table 1).

2.7. Histopathology Studies. The mice were sacrificed 24 hafter the last CS treatment.The representative skin specimenswere peeled and fixed in 10% formalin neutral bufferedsolution at least for 1 d to process paraffin sections. 5𝜇m thicksections were stained with Hematoxylin & Eosin (H&E) forroutine histology study. Besides, the sections were stainedwith Weigert’s resorcin fuchsin to represent elastic fibers andMasson’s trichromatic staining was used to reflect the densityof collagen fibers [19].

In slides of every H&E stained section (magnification,200x) hyperplastic epidermal layer was vertically measuredat 10 selected locations using an optical microscope (LeicaDMLB).

2.8. Measurements of Antioxidant Enzymes and Lipid Per-oxidation in the Skin. Sample preparation was according tocorresponding diagnostic kits. The skin tissue was homoge-nized with Ultra Turrax (T18 Basic, IKA) at 10,000 rpm for 20seconds in 9-fold volumes of cold normal saline to get the 10%homogenate, 0.15mL of which was taken out for the MDAassay.The remainder was centrifuged at 3,000 rpm for 20minat 4∘C to obtain supernatant used forCAT, SOD,GSH-Px, andprotein concentration measurements.

2.9. Determination of Inflammatory Factors and MMP-1 andMMP-3 in the Skin. Another skin tissue was processed inthe same way but in cold phosphate buffer solution andthen the total supernatant was applied for IL-1𝛽, IL-6, andTNF-𝛼, COX-2 and PGE

2, and MMP-1, MMP-3, and protein

concentration.

2.10. Determination of Total Collagen Content. Collagen con-tent can be converted fromHyp content, which was regardedas a characteristic amino acid of collagen, by multiplyingthe factor 7.46 [20]. About 100mg fresh skin tissues were

hydrolyzed in 6.0M hydrogen chloride for Hyp kits to revealthe total collagen con.

2.11. Analysis of UV Absorption Spectrum and Sun ProtectionFactor (SPF). CS aqueous extract was diluted to the concen-tration of 2mg/mLwith ethanol and filtered through 0.45𝜇mmicroporousmembrane for use. Full wavelengthwas set from200 nm to 800 nm for analyzing theUVabsorption spectrum.SPF is an important indicator to assess the photoprotectioneffect of sunscreen [21]. Ultraviolet spectrophotometry wasused to determine SPF. The calculation was based on formu-lation (1) [22]. Optimized range to obtain absorbance wasfrom 290 nm to 320 nm.Three doses of CS were, respectively,diluted 500 times with ethanol and filtered through 0.45𝜇mmicroporous membrane for determination. Determinationswere performed every 5 nm, repeated for 3 times at eachpoint. The ultraviolet spectrometry photometer (TechcompLtd., UV1000) and matching 1 cm quartz cuvettes were usedin the above detections:

SPFspectrophotometric = CF ×320

∑290

EE (ℷ) × I (ℷ) × Abs (ℷ) . (1)

EE (ℷ): erythemal effect spectrum; I (ℷ): solar intensityspectrum; Abs (ℷ): absorbance of sunscreen product; and CF:correction factor (=10). The values of EE × I were constantsdetermined by Sayre et al. [23].

2.12. Statistical Analysis. One-way analysis of variance(ANOVA) was used to detect group differences. A value of𝑝 < 0.05 was considered to be statistically significant. Allanalyses were performed using SPSS 17.0.

3. Results

3.1. Chemical Compositions of CS. The chemical composi-tions of CS aqueous extract were determined by HPLC-ESI-MS analysis, 10 components of which were identifiedas methyl rosmarinate, ethyl 3,4-dihydroxyphenyllactate,baicalein, danshensu, protocatechuic acid, p-hydroxybenzoicacid, rosmarinic acid, caffeic acid, ferulic acid, and 2𝛼-hydroxy-ursolic acid presented in Table 2 and Figure 1.

3.2. CS Alleviated UV-Induced Skin Lesions. The representa-tive photos of UV-induced skin lesions were shown in Fig-ure 2(a). After ten weeks, dorsal skins of mice in the MC andVC groups presented a coarse and corrugated appearance.Additionally, erythema, edema, and skin burns appearedfrequently in these two groups. The abnormally changedskin appearances proved that the photoaging model in ratswas successfully established, and the vehicle had no effectto ameliorate the UV-induced lesions. Contrarily, mice inthe SC group which had not been irradiated showed neitherwinkles nor lesions, proving that the shaving operation hadno macroscopic damage to skin. Compared to the VC group,the mice skin in the CS-H group presented much moresmooth appearance without observable lesions. Meanwhile,dorsal skins of mice in the CS-M group displayed a few


Table 2: HPLC-ESI-MS analysis of CS aqueous extract.

Number RT (time) Molecular weight Parent ion (𝑚/𝑧) Product ion (𝑚/𝑧) Compoundsa[M + H]+ [M −H]−

1 4.4 374.34 375.1 — 231, 213, 114 Methyl rosmarinate2 5.3 226 — 225.1 223, 179, 161, 87 Ethyl 3,4-dihydroxyphenyllactate3 5.7 270.24 — 269.1 109, 88, 61 Baicalein4 6.7 198.17 — 197.0 135, 123, 109, 73 Danshensu5 12.8 154.12 — 153.0 119, 108, 91 Protocatechuic acid6 21.5 138.12 — 137.0 93, 65 p-Hydroxybenzoic acid7 22.4 360.31 — 359.1 197, 179, 161, 135 Rosmarinic acid8 24.8 180.15 — 179.0 136, 135, 134 Caffeic acid9 40.5 194.18 — 193.1 165, 150, 78 Ferulic acid10 45.4 472.69 473.36 — 464, 416, 408, 360 2𝛼-Hydroxy-ursolic acidaAnalyzed by HPLC-ESI-MS analysis both in positive and in negative ionization modes.

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75Time (min)

1

10Inte

nsity

3.0e72.5e72.0e71.5e71.0e75.0e60.0e0

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9e68e67e66e65e64e63e62e61e60e0

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Figure 1: Total ion chromatorgraphies of CS aqueous extract by HPLC-ESI-MS/MS analysis. (a) Positive ion mode. (b) Negative ion mode.

wrinkles, whereas mice in the CS-L group exhibited shallowwrinkles and slight erythema.

Statistically, the visual scores of SC group were muchlower than that in both MC and VC groups from the thirdweek (Figure 2(b)). However, after seven weeks, the visualscores of mice skin in CS-H and CS-M groups were obviouslydecreased (both 𝑝 < 0.05, versus VC group), while theformation of wrinkles from the ninth week started to slowdown in the CS-L group. These results indicated that topicalapplication of CS could cure the UV-induced erythema andedema in a dose-dependent manner.

3.3. CS Reduced Epidermal Thickness. Epidermal thicknesscan be used to reflect photoaged skin, since it can result inskin roughness and winkles. As illustrated in Figure 3(b),no remarkable difference in epidermal thickness was foundbetween theNCand SCgroups aswell as between theMCandVCgroups, which implied that the shaving and the vehicle didnot affect this indicator. Moreover, the epidermal thicknessesof mice in MC and VC groups dramatically increased afterchronic UV exposure, which were 4.63 and 5.20 times ofSC mice (15.62𝜇m). However, the epidermal thickening wasmarkedly decreased to 35.58 𝜇m, 35.85 𝜇m, and 31.68 𝜇m inthe CS-L, CS-M, and CS-H groups, respectively (all 𝑝 <0.05 versus VC group). Besides, a result of dorsal skin-fold thickness measured by vernier caliper also supportedthe potential inhibitory effect of CS on skin thickening(Figure 2(c)).

3.4. CS Prevented UV-Induced Skin Structure Damage.Repeated UV irradiation led to abnormal histological alter-ations of the mice skin. Dorsal skins of the SC and NCgroups both manifested clear and complete skin structureswith thin layer of stratum corneum covering normal epi-dermis and wavy dermal-epidermal junction (DEJ) lines.In the superficial dermis, neither inflammatory infiltrationnor hemorrhages were observed (Figure 3(a)). Hair follicleswere distributed regularly and collagen bundles stained pinkinterweaved closely and arranged orderly (Figure 4(a)), whileelastic fibers stained deep purple were slender and branched(Figure 4(b)). After ten weeks, similar histopathologicalfeatures could be observed in MC and VC groups. In theepidermis, both of them showed epidermal hyperplasia andexcessive keratinization as well as obviously thickened stra-tum corneum. Additionally, DEJ lines turned to be flattened,which could decrease the surface contact area and then causefragile skin. In the dermis, large amounts of the collagenfiber bundles were disorganized and destructed companiedwith plenty fragmented elastic fibers. Moreover, skins of MCand VC groups presented hemorrhage and inflammatoryinfiltration in the entire dermis.

Nevertheless, CS especially at middle and high dosesobviously ameliorated this structure damage caused by UVlight. The skin features of CS-L group were much similarto the VC groups but presented more regular collagen bun-dles and less inflammatory infiltration. In CS-M and CS-Hgroups, skin structure damage got apparently improved (Fig-ure 3(a)). Firstly, the epidermal thickness was significantly


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Figure 2: CS alleviatedUV-induced skin lesions. (a) Visual appearance of different groups after the last treatment. (b)Visual scores of differenttreated groups. Data shown are the mean values ± SD (𝑛 = 9). #𝑝 < 0.05 compared with the SC group; ∗𝑝 < 0.05 compared with the VCgroup. (c) Skin-fold thickness of different groups. Data shown are the mean values (𝑛 = 9).

decreased and the DEJ lines were more curved. Secondly,abundant collagen bundles and elastic fibers were orderlydisplayed. Thirdly, diffused inflammation and hemorrhageswere absent in and underneath the dermis in the CS-H group.

3.5. CS Reduced UV-Induced Oxidative Stress. Compared tothe SC group, activities of antioxidant enzymes as SOD,CAT, and GSH-Px in the MC and VC groups were obviouslyreduced (𝑝 < 0.05). However, CS at high dose couldmaintainthe activity of SOD, CAT, and GSH-Px, which were increasedby 20.71%, 68.35%, and 43.93%, respectively (all 𝑝 < 0.05versus VC group). These results revealed that CS enhancedthe activities of antioxidant enzymes to suppress the UV-induced oxidative stress (Figure 5).

3.6. CS Decreased the Skin MDA Content. When comparedwith SC group, obvious increase of MDA level was found inMCandVCgroups (𝑝 < 0.05 versus SC group). Nevertheless,the MDA content in the CS-H group was notably decreased

by 35.11%, as compared with that in the VC group.Therefore,CS inhibited lipid oxidation by diminishing MDA and theeffect of the high dose of CS was much better compared tothe middle and low doses (Figure 5).

3.7. CS Suppressed Inflammatory Cytokines Production.Inflammatory cytokines (IL-1𝛽, IL-6, and TNF-𝛼) weresubstantially produced in the MC and VC groups (𝑝 < 0.05versus SC group). However, the production of IL-1𝛽, IL-6,and TNF-𝛼 in the CS treated groups especially in middledose and high dose was significantly suppressed (for all𝑝 < 0.05 versus VC group). The results showed that CSrestrained the generation of inflammatory cytokines inducedby UV irradiation (Figure 5).

3.8. CS Inhibited COX-2 to Hinder the Synthesis of PGE2. The

expressions of COX-2 and PGE2in theMC group were much

higher than these in the SC group (both 𝑝 < 0.05 versus SCgroup). However, the contents of COX-2 in CS groups were


HF

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Figure 3: CS reduced epidermal thickness. (a) Histological images of mice skin via H&E staining. Epidermal thickness was shown via thedouble-headed red arrows. NC group (100x) and SC group (100x) were representing a normal structure; HF, hair follicle; DEJ, dermal-epidermal junction; ED, epidermis; DR, dermis; and ST, subcutaneous tissue. MC group and VC group were showing similar abnormalstructure (100x); (A) SC, stratum corneum (100x); (B) hemorrhage (200x); (C) IF, inflammation infiltration (100x); CS-L group (100x); CS-Mgroup (100x); and CS-H group (100x). (b) Histograms of average epidermal thickness. #𝑝 < 0.05 compared with the SC group; ∗𝑝 < 0.05compared with the VC group.


NC SC MC

VC50𝜇m50𝜇m50𝜇m50𝜇m

50𝜇m50𝜇m50𝜇m

CS-L CS-M CS-H

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NC SC MC

VC CS-HCS-MCS-L

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Figure 4: (a) Collagen fibers ofmice skin viaWeigert’s staining (all photographsweremagnified at 100x). NC and SC group presented collagenfiber bundles of high content. MC and VC groups showed disordered and scattered collagen fiber bundles. (b) Elastic fibers of mice skin viaMasson’s trichromatic dyeing (all photographs were magnified at 400x). NC and SC group exhibited abundant organized elastic fibers; MCand VC groups showed ruined and fractured elastic fibers.

reversed to about normal levels. When treated with CS, thecontents of COX-2 were decreased by 30.49%, 25.40%, and29.60%, respectively, while the PGE

2expressions were lower

in a certain degree compared to that in the VC groups (all

𝑝 < 0.05 versus VC group).The results showed that CS couldinhibit the production of COX-2 to hinder the synthesis ofPGE2and thus ameliorate the skin lesions and algesia induced

by UV irradiation (Figure 5).


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Figure 5: CS inhibited UV-induced oxidative stress and LPO, suppressed the generation of inflammatory cytokines, downregulated MMP-1and MMP-3 levels, and prevented skin collagen from UV damage. Data shown are the mean values ± SD (𝑛 = 9). #𝑝 < 0.05 compared withthe SC group; ∗𝑝 < 0.05 compared with the VC group.

3.9. CS Enhanced Skin Collagen Content in Photoaged Mice.Collagen helps skin to be firmed and flexible and plays animportant role in constant cell metabolism [2]. After longtime of UV irradiation, the MC and VC groups showedobvious decrease in collagen content (both 𝑝 < 0.05 versusSC group). After being treated with CS the collagen contentswere evaluated (for all 𝑝 < 0.05 versus VC group). Theseresults manifested that CS could protect skin collagen fromUV damage. In Figure 4(a), the optical density of collagenbundles stained deep blue also confirmed the preservativeeffects of CS on collagen degradation (Figure 5).

3.10. CS Reduced Overexpression of MMPs. It is reported thatMMPs (especially MMP-1 and MMP-3) are responsible for

the degradation of skin collagen [5]. The contents of MMP-1and MMP-3 in the VC group were much higher (both 𝑝 <0.05 versus SC group). In the CS treated groups the contentsof MMP-3 were obviously decreased (all 𝑝 < 0.05 versus VCgroup). What is more, CS at high dose inhibited the lift ofMMP-1 (𝑝 < 0.05 versus VC group). These results suggestedthat CS could effectively reduce the overexpressions ofMMP-3, as well as MMP-1, thereby preventing the UV-inducedcollagen degradation (Figure 5).

3.11. UVAbsorption Spectrum and SPF of CS. TheUVabsorp-tion spectrum of CS was presented in Figure 6. The largestabsorption wavelength was 280 nm. SPF values of CS werelisted in Table 3, which were in positive correlation with dose.


Table 3: SPF values of CS aqueous extract at different doses.

Doses of CS (g/mL) SPF values0.9 3.92 ± 0.01

1.8 7.90 ± 0.02

3.6 16.21 ± 0.03

Abs

3.0000

2.0000

1.0000

0.0000200 400

280

600 800 (nm)

Figure 6: The UV absorption spectrum of CS aqueous extract(200 nm to 800 nm).

The SPF value in CS-H groupwas 2.05-fold to CS-M and 4.14-fold to CS-L group.

4. Discussion

Photoaging is a comprehensive consequence of exposure toUV irradiation, which is clinically characterized with sag-ging, coarseness, wrinkling, erythema, and dyspigmentation.Consistent with the previous studies, UV irradiation visiblyaccelerated wrinkle formation and increased skin thicknessin our study. However, CS had a tendency to alleviate theapparent lesions and slow down the progress of photoaging,confirmed by the fact that the formation of wrinkles andthe skin-fold thickness in the CS groups were apparentlylower than those in the VC group. The SPF values alsoconfirmed the photoprotection effects of CS, which were ina dose-dependent manner. Results of HPLC-ESI-MS analysisshowed that compounds as caffeic acid [24], ferulic acid [25],ursolic acid [26], p-hydroxybenzoic acid [27], rosmarinicacid [28], and protocatechuic acid [29] existed in CS. Thesecompounds had been reported to exhibit protective effectagainst UV irradiation in vitro via reducing oxidative stress.These active substances made the foundation of photoprotec-tive ability of CS.

Histological changes were characterized with increasedthickness of stratum corneum and excessively keratinizedepidermis after exposure to UV. In the dermis of photoagedskin, the collagen fiber bundles were tangled and destructedcompanied with elastic fibers dramatically degraded.The pri-mary mechanism of these abnormal histological alterationswas overproduction of MMPs, such as MMP-1 and MMP-3. MMP-1 (interstitial collagenase-1) breaks down fibrillar

collagens and elastin, while MMP-3 (stromelysin-1) degradeselastic fibers and other connective components, thereforeresulting in a loss of skin’s ability to resist stretching [1].In this study, we found that CS suppressed the increase ofMMP-1 andMMP-3 production and promoted the density ofcollagen and elastic fibers.These results indicated that CS canbe used as MMPs inhibition to remodel extracellular matrixstructures in the tissue, thereby alleviating UV-induced skindamage.

It has been reported that excessive MMPs resulted fromresultant accumulation of ROS and inflammation responses.The protection mechanism against ROS damage is antiox-idant defense system [30]. Intrinsic antioxidant enzymescould scavenge ROS and protect skin cells. SOD acceleratesthe reduction of O

2

− into O2and H

2O2, and CAT catalyzes

H2O2into O

2and H

2O [30]. SOD cooperated with CAT

in eliminating O2initiated ROS. In addition, GSH-Px also

breaks down H2O2with the substrate glutathione [30].

Although there are efficient antioxidant systems in skin,the excessive ROS induced by UV exposure exceeds theircapacity. Hsu et al. [10] had determined ROS scavengingand Trolox equivalent antioxidant capacity (TEAC) as well asOxygen Radical Absorbance Capacity (ORAC) assay of CSwater extract in vitro. The results of that study fully provedthe ROS scavenging potential and antioxidant property ofCS. In this study, CS especially at high dose could pro-tect antioxidant enzymes and reduce oxidative stress. Theseresults revealed that CS possessed antiphotoaging effect onthe foundation of scavenging free radical and enhancingantioxidant activities. Such antioxidant efficacy of CS in vivowas owed to phenolic acids in CS such as rosmarinic acid,caffeic acid, and danshensu. These phenolic acids not onlyexhibited both free radical scavenging activities to defendbody’s antioxidant mechanism but also stimulated tyrosinaseto expedite melanin production [24, 28, 31, 32].

What is more, lipid peroxidation (LPO) is initiated byROS on polyunsaturated fatty acids and aggravates LPOin membranes and cellular components and subsequentlycauses cell death and accumulation of abnormal proteins andcellular debris, finally leading to serious skin pathologies [33].LPO can be further decomposed to a number of reactive alde-hyde species such as MDA [34]. Therefore, MDA is usuallyused as a parameter to quantify LPO. However, CS coulddownregulate the accumulation of MDA in mice skin. Proto-catechuic acid and rosmarinic acid in CS have been reportedto have antilipid peroxidative properties throughmodulatingof cellular redox status with the upregulated expression ofantioxidant enzymes, including heme oxygenase-1, SOD,and CAT. Therefore, CS could protect the UV-induced skindamage by depressing LPO [31, 35].

Furthermore, a characteristic response of keratinocytes tochronic UV irradiation is the oxidation of the arachidonicacid in cell membranes, which is catalyzed by the enzymeCOX-2. COX-2 can result in the formation of oxidationproducts such as PGE

2, which are additional mediators of

inflammation reaction. The expression of COX-2 has beendocumented in inflammation response, while intradermalPGE2is hyperalgesic in the peripheral nervous system. PGE

2

acts as a potent vasodilator and synergistically with other


mediators increases vascular permeability and other inflam-matory markers such as erythema, edema, and hyperplasticepithelial responses [6]. Expression of COX-2 is upregulatedby ROS to stimulate the inflammation process. However, CSsignificantly reduced the contents of COX-2 and PGE

2in

mice skins. It could be due to the chemical compounds in CSsuch as caffeic acid phenethyl ester [36], protocatechuic acid[37], rosmarinic acid [28], and baicalein [38] to confer anti-inflammatory properties.

Exposure to UV light usually motivates inflammation.The regulatory mechanisms include releasing of upstreamproinflammatory cytokines such as TNF-𝛼 from keratino-cytes. UV irradiation stimulates TNF-𝛼 synthesis and releasesit into blood stream, which activates downstream cytokinessuch as IL-3, IL-1𝛽, and IL-6. These inflammatory fac-tors mediate the growth of keratinocytes in epidermisand lead to epidermal hyperplasia [1]. Consistent with theprevious research, the contents of IL-1𝛽, IL-6, and TNF-𝛼 were sharply lifted after exposure to UV in our study.However, when treated with CS, IL-1𝛽, IL-6, and TNF-𝛼contents were remarkably reduced to about normal level.The inflammatory infiltration could not be observed inthe H&E sections of CS-H group, which was proved bysmooth skin appearance (Figure 2(a)). Combined withresults of HPLC-ESI-MS/MS analysis, chemical compo-nents in CS such as baicalein and protocatechuic alde-hyde were crucial for its distinguished anti-inflammatoryactivity through inhibition of COX-2 gene expression byblockade of C/EBPbeta DNA binding activity [38] and theTNF-𝛼-activated NF-𝜅B and AP-1 DNA binding activities[39].

In conclusion, our research for the first time applied CSto the mice skin and demonstrated that CS could postponeexogenous senescence via defending antioxidant activitiesand suppressing inflammatory response. It indicated that CSis a potential agent for antiphotoaging cosmetics.

Competing Interests

The authors declare no conflict of interests.

Authors’ Contributions

Lan Wang and Xie Zhang contributed equally to this work.

Acknowledgments

This work was supported by grants fromHong Kong, Macao,and Taiwan Science & Technology Cooperation Programof China (no. 2014DFH30010), Guangdong InternationalCooperation Project (no. 2013508102016), and Science andTechnology Planning Project of Guangdong Province, China(no. 2013B090600007). Guangdong Provincial Key Labora-tory of New Chinese Medicinals Development and Research,Guangzhou University of Chinese Medicine, Guangzhou510006, China, is acknowledged.

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